title: Coupling efficiency of rhodopsin and transducin in bicelles.
abstract: G protein coupled receptors (GPCRs) can be activated by various extracellular stimuli, including hormones, peptides, odorants, neurotransmitters, nucleotides, or light. After activation, receptors interact with heterotrimeric G proteins and catalyze GDP release from the Gα subunit, the rate limiting step in G protein activation, to form a high affinity nucleotide-free GPCR-G protein complex. In vivo, subsequent GTP binding reduces affinity of the Gα protein for the activated receptor. In this study, we investigated the biochemical and structural characteristics of the prototypical GPCR, rhodopsin, and its signaling partner, transducin (G(t)), in bicelles to better understand the effects of membrane composition on high affinity complex formation, stability, and receptor mediated nucleotide release. Our results demonstrate that the high-affinity complex (rhodopsin-G(t)(empty)) forms more readily and has dramatically increased stability when rhodopsin is integrated into bicelles of a defined composition. We increased the half-life of functional complex to 1 week in the presence of negatively charged phospholipids. These data suggest that a membrane-like structure is an important contributor to the formation and stability of functional receptor-G protein complexes and can extend the range of studies that investigate properties of these complexes.
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, United States.
authors.forename: Ali I
authors.initials: AI
authors.lastname: Kaya
authors.forename: Tarjani M
authors.initials: TM
authors.lastname: Thaker
authors.forename: Anita M
authors.initials: AM
authors.lastname: Preininger
authors.forename: T M
authors.initials: TM
authors.lastname: Iverson
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Hamm
mesh_terms: D055162:Biocatalysis; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D008027:Light; D008823:Micelles; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D012243:Rhodopsin / Q000378:metabolism*; D012542:Scattering, Radiation; D013696:Temperature; D015293:Transducin / Q000378:metabolism*
chemical_list: D003902:Detergents; D008823:Micelles; D009711:Nucleotides; D012243:Rhodopsin; D015293:Transducin